Natural killer (NK) cells are bone marrow derived lymphocytes that constitute a key frontline defense against a range of hazardous conditions including viral infection and tumor transformation1. Although NK cells can kill target cells spontaneously without prior stimulation, a delicate balance between inhibitory and activating signals tightly regulates their activation. NK cells express a family of inhibitory and activating receptors that recognize major histocompatibility complex (MHC) class I molecules1. Engagement of these receptors results in the transduction of inhibitory signals that, under normal physiological conditions, predominate over those mediated via activating receptors1. This insures that healthy cells, expressing adequate amounts of MHC class I, will be protected from NK cells attack, while cells that have lost normal expression of MHC class I molecules, often resulting from viral infection or cell transformation, will be destroyed2.
This concept, however, accounts only in part for the regulation of activation, and non-MHC ligands also control NK-mediated killing3. In addition, certain NK-susceptible target cells express normal MHC class I repertoire, while some MHC-negative cells are resistant to NK attack4. It has now become evident that activation of NK cells requires more than just the absence of inhibitory signals and that in order to tip the balance in favor of NK cells activation, target cells must express ligands specific for activating receptors.
A significant breakthrough in the understanding of specific activation of NK cells was achieved following the recent identification of three novel NK specific triggering receptors collectively termed natural cytotoxic receptors (NCR). The NCRs, which include NKp46, NKp44 and NKp30, belong to the Ig superfamily but share no homology with each other and only a low degree of identity with any known human molecules5,6,7. Upon engagement, NCRs transduce activating signals through the association with various adaptor molecules (including the DAP12 and zeta chain proteins) that carry immunoreceptor tyrosine-based activation motifs (ITAM) in their cytoplasmatic tail, allowing activation via the src-kinase and syc-signaling pathway5,7,8. All of these NCRs are capable of mediating direct killing of tumor and virus-infected cells and are specific for non-MHC ligands. NKp46 and NKp30 are present exclusively on NK cells, whether resting or activated, while NKp44 is expressed specifically on activated NK cells.
The most distinctive role of the NCRs in NK cells activity has been attributed to their involvement in recognition and killing of tumor cells. This has become evident by the ability of anti-NCR monoclonal antibodies to block NK-mediated killing of most tumor lines4-7,9 and by the strict correlation that exists between the density of NCRs expression on NK cells and their ability to kill tumor targets9. More recently, the importance of NCRs in vivo was illustrated in acute myeloid leukemia (AML) patients expressing insufficient amount of either NCR or NCR ligands, thereby rendering the leukemia cells resistant to NK cytotoxicity10.
While NCRs have been implicated most conclusively in immunity against transformed cells, there is evidence they may also contribute to defense against pathogens1,2,11,12. This notion is supported by the fact that NK deficient individuals suffer from a range of recurrent diseases, especially viral infections12. Recently, the inventors and coworkers have demonstrated that soluble NKp46- and NKp44-immunoglobulin (Ig) fusion proteins, but not an NKp30-Ig fusion protein, specifically bind to hemagglutinin of influenza virus and to hemagglutinin-neuraminidase of Sendai virus13-15. This interaction is functional and can mediate an enhanced killing of infected cells. This enhanced killing can be abolished by antibodies that block either the HA or the lysis receptors NKp46 and NKp4413-15.
The human NKp46 receptor has multiple isoforms including the currently known isoforms: Isoform a (Accession No CAA04714; SEQ ID NO:1); Isoform b (Accession No. CAA06872) Isoform c (Accession No. CAA06873) Isoform d (Accession No. CAA06874). In general the NKp46 receptor comprises two extracellular Ig-like domains of the C2 type, a transmembrane portion and an intracellular segment. The extracellular portion of NKp46 comprises a D1 domain, designated NKp46D1 (comprising residues 22-120 of the mature full length protein of isoform a) a D2 domain, designated NKp46D2, comprising 134 amino acid residues (residues 121-254 of the full length receptor of isoform a; SEQ ID NO:2).
The human NKp44 receptor (accession No CAC09453; SEQ ID NO:4) comprises one extracellular portion designated herein NKp44D (residues 22-135 of the full length receptor; SEQ ID NO:5), comprising a single globular domain and a tail (residues 136-190).
WO 02/08287 of the present inventors discloses NK receptor fusion proteins in which the extracellular portion of the various NK receptors is conjugated to an active segment comprising an immunoglobulin (Ig), a cytotoxic moiety or an imaging moiety. WO 02/08287 further discloses that the NK receptor fusion proteins exhibit specific interaction with tumor cells and viral-infected cells in vitro.
There is an unmet need for characterization of the molecular features or epitopes involved in killing of viral infected and tumor cells. The present invention discloses peptides derived from the amino acid sequences of NKp44 and NKp46, and essential epitopes therein that are crucial for the binding of these receptors to viral-infected and tumor cells.